1. Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen presenting cells which critically regulates cell-mediated immunity. Because of its crucial function during immune responses, IL-12 production is stringently regulated, in part through transcriptional control of its p35 subunit which requires the differentiative effects of interferon-gamma for expression. To determine whether post-transcriptional aspects of IL-12 production might be regulated, we examined intracellular protein processing of each subunit. We demonstrated that p40 is processed in a manner consistent with the signal peptide/signal peptidase model but that processing of p35 is not. Instead processing of the p35 preprotein required two sequential cleavages to generate the secreted form of the protein. Initial cleavage of p35 occurred within the hydrophobic domain of the signal peptide and was accompanied by, but not dependent upon, partial glycosylation within the ER. Cleavage to release the amino terminus of the secreted form of the protein appeared to be a rapid, transient process with no detectable cellular accumulation, and appeared concomitant with additional complex glycosylation. The second cleavage site was shown by mutational analysis to conform to a monobasic protease recognition sequence. Proteases recognizing monobasic cleavage sites appear to have specific subcellular distributions (including the Golgi compartments) and different enzymes have been reported for every preprotein examined, raising the concern that not all tissues may be able to process p35 by this pathway. Glycosylation appeared to be requisite for p35 but not for p40 secretion. 2. Our current understanding of the biology of IL-12 has been limited to studies which can be performed using circulating cells of the hematopoietic lineage. Without direct examination, the results of these studies have been assumed to apply to tissue-resident cells which constitute a first-line defense during many types of innate and acquired immune responses. We developed a model of innate immunity to understand better the contributions tissue-resident cells make towards IL-12-mediated host defense mechanisms and to address safety concerns for the use of IL-12 gene therapies. Using this model we found evidence for a previously unrecognized third IL-12 receptor chain (provisionally named IL-12Rbeta3) which is induced following chronic, but not short-term, IL-4 treatment. This new chain appears to be expressed in the absence of IL12 Rbeta1 and to complement the IL-12Rbeta2 subunit which has a low binding affinity and does not signal by itself in the absence of additional subunits. Studies are currently underway to molecular clone, sequence, and characterize this new molecule.